Immunoassay with novel labeled drug hapten analogues

ABSTRACT

The invention is directed to labeled drug hapten analogues comprising: 
     (A) a label, of the type used in immunoassays, having an amine or sulfhydryl group; 
     (B) a drug hapten nucleus selected from barbiturates or hydantoins and 
     (C) a linking chain linking the 3-position of the drug hapten nucleus to the label through a carbonyl bridge.

This is a continuation of application Ser. No. 851,436, filed Mar. 16,1992, now abandoned, which is a continuation of application Ser. No.712,328, filed Jun. 7, 1991, now abandoned.

FIELD OF THE INVENTION

This invention relates to clinical chemistry particularly immunoassays.

BACKGROUND OF THE INVENTION

Immunoassays, which take advantage of natural immunological reactions,have found wide-spread use as analytical techniques in clinicalchemistry. Because of the specificity of the reactions, they areparticularly advantageous in quantifying biological analytes that arepresent in very low concentration in biological fluids. Such analytesinclude, for example, antibodies, therapeutic drugs, narcotics, enzymes,hormones, proteins, etc.

In competitive binding immunoassays, a labeled ligand, includingimmunocompetent derivatives and analogs of the ligand is placed incompetition with unlabeled ligand for reaction with a fixed amount ofthe appropriate binding material (called a receptor herein). Unknownconcentrations of the ligand can be determined from the measured signalof either the bound or unbound (i.e. free) labeled ligand. The reactionproceeds as follows:

ligand+labeled ligand+receptor→ligand-receptor+labeled ligand-receptor.

Conventional labels include radioactive tags, enzymes, chromophores,fluorophores, stable free radicals, and enzyme cofactors, inhibitors andallosteric effectors.

Consistent with the foregoing an immunoassay for drugs, such asphenobarbital and phenytoin, in serum can be based on competition of anenzyme labeled analogue of such drug haptens with the drug in thepatient serum for immobilized antibody binding sites.

Specific requirements for the labeled drug hapten analogues (hereaftersometimes LDH) include: 1) at least 65% of the LDH can be bound byexcess immobilized antibody; 2) affinity of the LDH for immobilizedantibody is such that competition of a fixed amount of LDH with the drugoccurs in a therapeutically relevant drug concentration range; and 3)stability of the LDH against hydrolysis of its enzyme label understorage conditions.

Requirements imposed on the drug hapten analogue include: 1)accessibility of the analogue to the immobilized antibody followingconjugation with the enzyme label; 2) specific recognition of the LDHanalogue by the antibody to the drug; and 3) sufficient reactivity ofthe analogue with the enzyme label, either directly or followingactivation of the enzyme or the analogue, under conditions that do notadversely affect enzyme activity.

Glucose oxidase (GOD) and alkaline phosphatase (ALP) enzyme labelscoupled to phenobarbital and phenytoin hapten analogues disclosed inU.S. Pat. 4,752,568 gave adequate enzyme labeled analogues forconducting effective competitive immunoassays in the desired format.

The problem is that the labeled phenobarbital and phenytoin analoguesdisclosed in the above patent were unsatisfactory for conductingcompetitive immunoassays when the enzyme horseradish peroxidase (HRP)was used as the label. The coupling reactions between such analogues andHRP were both slow and incomplete. Moreover phenobarbital and phenytoinHRP labels were bound very weakly so that much higher concentrations oflabel or antibody binding sites would be required to give a readablesignal.

SUMMARY OF THE INVENTION

The present invention provides an immunoassay method for a drugcomprising the steps of:

A. contacting a liquid sample, containing the drug or a derivativethereof, with a labeled analogue of the drug or drug derivative in thepresence of antibodies for the drug under conditions that promote theformation of antibody-drug immunocomplexes; and

B. determining the quantity of the drug in the liquid by measuring boundor unbound labeled drug analogue; characterized in that the labeled druganalogue comprises:

(i) a label, of the type used in immunoassays, having an amine orsulfhydryl group;

(ii) a drug nucleus selected from a hydantoin nucleus or a barbituratenucleus and

(iii) a linking chain linking the 3-position of the barbiturate orhydantoin nucleus to the label through a carbonyl bridge; wherein thelinking chain has about 5 to 40 atoms, consisting of (1) C₁ to C₁₀alkylene groups, (2) phenylene groups, and (3) 5 to 7 memberedheterocyclic rings joined into the linking group through ring nitrogenatoms, wherein said groups and rings are bonded to each other throughchemical groups selected from (a) esters, including thioesters ##STR1##where Z is O or S; (b) amides, ##STR2## (c) hetero atoms selected from--O--, --S--, and --NR--; wherein R represents C₁ to C₆ alkyl; and (d)carbonyl, with the proviso that the linking group is other than only aderivative of a saturated or unsaturated monocarboxylic acid having fromto 2 to 12 carbon atoms.

The labeled drug hapten analogues useful in the above method includethose that conform to the structure: ##STR3## wherein A represents ahydantoin nucleus of the structure ##STR4## or a phenobarbitalbarbiturate nucleus of the structure ##STR5## R¹ each independentlyrepresents hydrogen, alkyl of 1 to 10 carbon atoms, unsubstituted orsubstituted phenyl;

R², R⁴, R⁵, and R⁶, each independently represents C₁ to C₁₀ alkylene orsuch alkylene groups interrupted with at least one or more ester groups,amide groups, --O--, --S--, and --NR--;

R³ represents C₁ to C₃ alkylene;

Z represents --O--, --S--, and --NR--, wherein R represents hydrogen orC₁ to C₆ alkyl, e.g. methyl propyl and hexyl;

m is 0, 1, or 2;

n is 0, 1, or 2;

m+n>0; and

the total number of atoms comprised in m, n and R² is 5 to 40;

LABEL is an enzyme;

and further provided that (i) at least one of the R¹ groups issubstituted or unsubstituted phenyl; (ii) one of R⁴, R⁵, and R⁶ can bephenylene; (iii) the bracketed components of structure I can appeartherein in any order and (iv) the linking group is other than aderivative of a saturated or unsaturated monocarboxylic acid having fromto 2 to 12 carbon atoms.

At least 65% of the labeled drug hapten analogues can be bound by anexcess of immobilized antibodies to the drugs. The labeled analogueswith extended linkers, particularly those having amide bonds in thelinking chain are similarly bound by all the immobilized antibody typesused in reducing this invention to practice. Also the derivatives havingthe amide in the extended linker are very stable against hydrolysis.

DETAILS OF THE INVENTION

The drug hapten analogues useful in the immunoassay provided by thepresent invention are those that comprise:

(a) an active ester group, such as a succinimidoxycarbonyl;

(b) a nucleus selected from hydantoin or barbiturate derivatives; and

(c) a linking chain linking the active ester group to the hydantoin orbarbiturate nucleus; wherein the linking chain is as previously defined.

More specifically, the preferred new drug hapten analgues useful in thisinvention are those conforming to the structure: ##STR6## wherein Arepresents a hydantoin nucleus of the ##STR7## or a barbiturate nucleusof the structure ##STR8## R¹, R², R³, R⁴, R⁵, R⁶, Z, m, and n and theprovisos related thereto are as previously defined, and

R⁷ is an ethylene or o-phenylene group. These new drug hapten analoguesreact with HRP, and other enzymes such as GOD and ALP, faster and morecompletely, than prior art analogues (b) form covalent bonds with suchenzymes without adverse effect on enzyme activity, and (c) the resultingLDH analogues are more readily recognized and tightly bound byantibodies to such drugs.

Hydantoin Drug Hapten Analogues

The novel hydantoin drug hapten analogues are disclosed in copendingU.S. patent application Ser. No. 712,329 filed 7 Jun. 1991, abandoned infavor of CIP U.S. Ser. No. 07/851,435 filed Mar. 16, 1992, the same dateas the present case.

Several advantages are realized by use of the above hydantoinderivatives. First, it was found that the active esters of thesehydantoin derivatives having short linking chains between the hydantoinnucleus and the active ester group were sufficiently reactive with HRPto prepare an acceptable enzyme label for use with some immobilizedantibodies. Derivatives with longer linker groups (R² plus the bracketedgroups) of 8 to 20 atoms between the active ester group and thehydantoin nucleus gave labels that could be bound by all immobilizedantibodies tested. Linking chains in which each Z is --NR-- which withthe adjacent carbonyl forms an amide group, are particularly useful inthat hydantoin derivatives containing such chains are more resistant tohydrolysis than chains wherein Z is --0-- or --S-- so that with theadjacent carbonyl it forms an ester group.

Hydantoin analogue Preparatory Examples

The hydantoin analogues can be made according to the followingpreparations in which phenytoin analogues, a subclass of hydantoincompounds, are made.

1. Preparation of HD1,5,5-Diphenyl-3-{4-[2-(3-succinimidoxycarbonylpropionyloxy)ethylaminocarbonyl]butyl}hydantoin.

Step 1: preparation of5,5-Diphenyl-3-[4-(2-hydroxyethylaminocarbonyl)butyl]hydantoin.

Part A: First the Acid Chloride is prepared.

A mixture of 3-(4-carboxybutyl)-5,5-diphenyl-2,4-imidazolidinedione(3.52 g, 0.01 mole) thionyl chloride (20 mL), N,N-dimethylformamide (2drops) and chloroform (50 mL) was stirred at room temperature for 3hours. The solvent was removed on a rotary evaporator in vacuo, and thisproduct was used directly in the next Part B.

Part B: The Acid Chloride is reacted with Ethanolamine.

The acid chloride in chloroform (50 mL) was added dropwise over 15minutes to a mixture of ethanolamine (1.2 g, 0.02 mole) andtriethylamine (2.4 g, 0.024 mole) in chloroform (100 mL). The mixturewas then heated to 60° C. for 2 hours and stirred to room temperaturefor 1 hour. The solution was then washed with 5% hydrochloric acid(2×100 mL), washed with saturated sodium bicarbonate solution (100 mL),dried over anhydrous magnesium sulfate, filtered, and the solventremoved on a rotary evaporator. The filtrate was then chromatographedusing an aluminum oxide column to give material (3.0 g) showing one spoton TLC. This material was used directly in the next preparation.

Step 2: preparation of3-{4-[2-(3-Carboxypropionyloxy)ethylaminocarbonyl]butyl}-5,5-diphenylhydantoin.

The hydroxy compound of Step 1 (3.0 g, 0.0075 mole), succinic anhydride(1.0 g, 0.01 mole), and dimethylaminopyridine (0.9 g, 0.0075 mole) inchloroform (100 mL) were heated at 50°-60° C. for 4 hours and allowed tocool to room temperature over the weekend.

Dichloromethane (300 mL) was added, and the mixture was washed with 5%hydrochloric acid solution (3×100 mL), washed with saturated sodiumchloride solution (100 mL), dried over anhydrous magnesium sulfate,filtered, and the solvent removed to give a material that gave one spoton TLC.

Step 3: preparation of HD 1:5,5-Diphenyl-3-{4-[2-(3-succinimidoxycarbonylpropionyloxy)ethylaminocarbonyl]butyl}hydantoin.##STR9##

A mixture of the acid from Step 2 (3.0 g, 0.006 mole),N,N'-dicyclohexylcarbodiimide (1.5 g, 0.007 mole), andN-hydroxysuccinimide (0.7 g, 0.006 mole) in chloroform (80 mL) wasstirred at room temperature for 20 hours. The mixture was filtered, andthe filtrate was concentrated on a rotary evaporator in vacuo. Theresidue was then chromatographed using silica to give 1.3 g (40% yield).

Anal. calc. for C_(3O) H₃₂ N₄ O₉ : C, 60.8; H, 5.44; N, 9.45. Found: C59.6; H, 5.51, N, 8.91

2. Preparation of HD 2:5,5-Diphenyl-3-{4-[4-(3-succinimidoxycarbonylpropionyl)-1-piperazinylcarbonyl]butyl}-2,4-imidazolidinedione

Step 1: preparation of5,5-Diphenyl-3-(1-piperazinylcarbonylbutyl)hydantoin.

Part A: First,3-[4-(4-Benzyloxycarbonyl-piperazinylcarbonyl)butyl]-5,5-diphenyl-2,4-imidazolidinedionewas prepared.

The acid chloride prepared as described in the preparation of HD 1,supra, Part A (0.01 mole) was added dropwise over 15 minutes to amixture of benzyl 1-piperazinecarboxylate (2.4 g, 0.011 mole) andtriethylamine (2.0 g, 0.02 mole) in chloroform (50 mL). This mixture wasstirred at room temperature overnight, and dichloromethane (300 mL) wasadded. The mixture was washed with 5% hydrochloric acid (2×100 mL),washed with dilute sodium carbonate solution (100 mL), washed withsaturated sodium chloride solution (100 mL), dried over anhydrousmagnesium sulfate solution, filtered, and the solvent removed on arotary evaporator in vacuo. The filtrate was then chromatographed togive an oil, 4.3 g (78% yield) which was used directly in the next step.

Part B: The protected amine of Part A (4.8 g, 0,008 mole) and 30-35%hydrogen bromide acetic acid solution (25 mL) was allowed to stir atroom temperature for 1.5 hours. This mixture was then poured intodiethyl ether (1 L), and the oil which separated was triturated withfresh portions of ether (3×1 L). The oil was dissolved in 10% aqueoussodium hydroxide solution (pH=14) and the aqueous solution extractedwith dichloromethane (4×100 mL). The combined organic solution waswashed with saturated sodium chloride solution (150 mL), dried overanhydrous magnesium sulfate, filtered, and the solvent removed in arotary evaporator in vacuo. The filtrate solidifies to give a whitesolid (2.6 g, 77% yield). This material was used directly in the nextstep.

Step 2: preparation of3-{4-[4-(3-Carboxypropionyl)-1-piperazinylcarbonyl]butyl}-5,5-diphenyl-2,4-imidazolidinedione.

A mixture of the amine from Preparation 7 (2.1 g, 0.005 mole) andsuccinic anhydride (0.54 g, 0.0054 mole) in chloroform (15 mL) washeated for 30 minutes at 50°-60° C. and allowed to stand at ambienttemperature for 20 hours. Dichloromethane (150 mL) was added, and themixture was washed with 5% hydrochloric acid (2×100 mL), saturatedsodium chloride solution (100 mL), dried over anhydrous magnesiumsulfate, filtered, and the solvent removed on a rotary evaporator invacuo to give a white solid, 2.5 g (95%) which was used directly in thenext step.

Step 3: preparation of HD 2:5,5-Diphenyl-3-{4-[4-(3-succinimidoxycarbonylpropionyl)-1-piperazinylcarbonyl]butyl}-2,4-imidazolidinedione.##STR10##

A mixture of the acid from step 2 (1.56 g, 0.003 mole),N,N'-dicyclohexylcarbodiimide (0.64 g, 0.003 mole), andN-hydroxysuccinimide (0.36 g, 0.003 mole) in chloroform (40 mL) wasstirred at room temperature over the weekend. The mixture was filteredand the solvent removed from the filtrate on a rotary evaporator invacuo to give 1.9 g (100% yield). The solid was chromatographed, and theproduct fraction was dissolved in dichloromethane (200 mL), washed withdilute sodium carbonate solution (2×100 mL), dried over anhydrousmagnesium sulfate, filtered, and the solvent removed on a rotaryevaporator to give a white solid which gives one spot on TLC.

3. Preparation of HD3,5,5-Diphenyl-3-{4-[6-(3-succinimidoxycarbonylpropionamido)hexylaminocarbonyl]butyl}-2,4-imidazolidinedione.

Step 1: preparation of3-[4-(6-Aminohexylaminocarbonyl)butyl]-5,5-diphenyl-2,4-imidazolidinedione.

Part A: preparation of3-[4-(6-Benzyloxycarbonylaminohexylaminocarbonyl)butyl]-5,5-diphenyl-2,4-imidazolidinedione.

The acid chloride prepared as an intermediate in the preparation of HD 1was treated with N-benzyloxycarbonyl-1,6-hexanediamine by the proceduresdescribed in step 1 in the preparation of HD 2, to give 7.5 g, 85%yield, of the protected amine.

Part B: The protected amine of Part A was treated with hydrobromicacid-acetic acid by the procedures of Step 1, Part B in the preparationof HD 2, to give the free amine which was used in step 3 withoutpurification.

Step 3: preparation of3-{4-[6-(3-Carboxy-propionamido)hexylaminocarbonyl]butyl)-5,5-diphenyl-2,4-imidazolidinedione.

This compound was prepared using the same procedures as step 2 of the HD2 preparation.

Step 4: preparation of HD 3:5,5-Diphenyl-3-{4-[6-(3-succinimidoxycarbonyl-propionamido)hexylaminocarbonyl]butyl}-2,4-imidazolidinedione.##STR11##

This material was prepared using the procedures of step 3 in thepreparation of HD 2 to give 2.6 g (80% yield), mpt 133°-134° C. Anal.Calc. for C₃₄ H₄₁ N₅ O₈ : C, 63.05; H, 6.38; N. 10.81. Found: C, 62.91;H, 6.41; N, 10.69.

Barbiturate Drug Analogues

The following preparatory examples 4 to 8 illustrate the preparation ofthe barbiturate drug hapten analogues for phenobarbital. The analoguesare generally prepared by (1) condensing a barbiturate derivative such,as phenobarbital, with an omegahaloalkanecarboxylate ester, (2)saponifying the ester to the corresponding acid, (3) conversion of theacid to the corresponding acid chloride and (4) condensation of the acidchloride with N-hydroxysuccinimide, or to further lengthen the linkingchain, with a diamine, diol, or aminoalcohol having one of the amine orhydroxy groups blocked, (5) deblocking, condensation with a dicarboxylicacid such as succinic acid, and then condensation with theN-hydroxysuccinimide to produce the analogue.

If desired, the condensation with a half-blocked diamine, diol, oraminoalcohol, and then another diacid can be repeated once or twice moreto further lengthen the linking chain. However, the same can beaccomplished with fewer steps by using longer chained diacids, diols,diamines, amino alcohols, or haloalkanecarboxylate esters.

4. Preparation of PB 1:5-Ethyl-5-phenyl-1-{4-[4-(3-Succinimidoxycarbonylpropionyl)-1-piperazinyl-carbonyl]butyl}-2,4,6(IH,3H,5H)pyrimidinetrione

Step 1: Preparation of5-ethyl-6-hydroxy-3-(4-methoxycarbonylbutyl)-5-phenyl-2,4(3H,5H)-pyrimidinedione

A mixture of phenobarbital (46.5 g, 0.2 mole) and tetrabutylammoniumhydroxide (500 mL, 0.2 mole of 0.4M in water) in dichloromethane (500mL) was prepared and to it was added methyl 5-bromovalerate (39.0 g, 0.2mole). The reaction mixture was stirred vigorously overnight (20 hrs).To this mixture was added saturated sodium chloride solution (100 mL),the organic layer was separated, and the aqueous solution was washedwith dichloromethane (2×100 mL). The combined organic solution waswashed with saturated sodium chloride solution (100 mL), dried overanhydrous MgSO₄, filtered, and the solvent removed.

Step 2: Preparation of3-(4-Carboxybutyl)-5-ethyl-6-hydroxy-5-phenyl-2,4(3H,5H)-pyrimidinedione

The 5-ethyl-6-hydroxy-3-(4-methoxy-carbonylbutyl)-5-phenyl-2,4(3H,5H)-pyrimidinedione ester (54.0 g, 0.156 mole) of step 1 in dioxane(500 mL), concentrated hydrochloric acid (55 mL), and water (55 mL) washeated at reflux for 4 hrs and at room temperature overnight. Thedioxane was removed under reduced pressure, and saturated sodiumchloride solution (250 mL) and dichloromethane (400 mL) were added tothe residue. The organic layer was separated, and the aqueous solutionwas extracted with dichloromethane (3×150 mL). The combined organicsolutions were washed with saturated sodium chloride solution (200 mL),dried over anhydrous MgSO₄, filtered, and the solvent removed. To theresidue was added diethyl ether, and the mixture was placed in a freezerat -16° C. over the weekend, and then filtered.

Step 3: Preparation of1-(4-chlorocarbonyl-butyl)-5-ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione

A mixture of the acid (6.6 g, 0.2 mole) from preparation 2, thionylchloride (50 mL), N,N dimethylformamide (2 drops), and chloroform (80mL) was stirred at room temperature for 1.5 hrs. The solvent was removedon a rotary evaporator in vacuo, and this product was used directly inthe next step 4.

Step 4: Preparation of1-[4-(4-Benzyloxy-carbonyl-1-piperazinylcarbonyl)butyl]-5-ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione

The acid chloride of step 3 (0.2 mole) in chloroform (75 mL) was addeddropwise over 15 minutes to a mixture of benzyl 1-piperazinecarboxylate(6.0 g, 0.030 mole) and triethylamine (4.0 g, 0.04 mole) in chloroform(100 mL). This mixture was stirred at room temperature for 20 hrs, anddichloromethane was then added (300 mL). The mixture was washed with 10%hydrochloric acid solution (3×100 mL), washed with saturated sodiumchloride solution (100 mL), dried over anhydrous magnesium sulfate,filtered, and the solvent removed. The residue was then chromatographedon SiO₂ to give a solid.

Step 5: Preparation of5-Ethyl-5-phenyl-1-[4-(1-piperazinylcarbonyl)butyl]-2,4,6-(1H,3H,5H)pyrimidinetrioneHydrobromide

The protected amine from preparation 4 (6.5 g, 0.012 mole) and 30-35%hydrogen bromide-acetic acid solution (30 mL) was allowed to stir atroom temperature for 1.5 hrs. The mixture was then poured into ethylacetate (2 L), stirred for 1 hr, filtered, and the solid washed with 500mL ethyl acetate.

Step 6: Preparation of1-{4-[4-(3-Carboxypropionyl)-1-piperazinyl-carbonyl]butyl}-5-ethyl-5phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione

The amine of step 5 (4.8 g, 0.01 mole), succinic anhydride (1.2 g, 0.012mole), and triethylamine (2.2 g, 0.02 mole) in chloroform (150 mL) wereheated for 30 min at 50°-60° C. (hot water) and allowed to stir atambient temperature for 16 hrs. Dichloromethane (200 mL) was added, themixture was washed with 10% hydrochloric acid solution (3×100 mL).saturated sodium chloride solution (100 mL), dried over anhydrousmagnesium sulfate, filtered, and the solvent removed on a rotaryevaporator in vacuo to give a white solid, 3.3 g (66%). This materialwas chromatographed using a SiO₂ column to give a white solid.

Step 7: Preparation of5-Ethyl-5-phenyl-1-{4-[4-(3-Succinimidoxycarbonylpropionyl)-1-piperazinylcarbonyl]butyl}-2,4,6(1H,3H,5H)pyrimidinetrione##STR12##

A mixture of the acid from step 6 (3.4 g, 0.007 mole),N,N'-dicyclohexylcarbodiimide (1.6 g, 0.008 mole), andN-hydroxysuccinimide (1.0 g, 0.008 mole) in chloroform (75 mL) wasstirred at room temperature for 20 hrs. The mixture was filtered, andethyl acetate (100 mL) was added. The organic solution was washed withwater (2×100 mL), saturated sodium chloride solution (50 mL), dried overanhydrous magnesium sulfate, filtered, and the solvent removed on arotary evaporator under vacuo. A portion of the solid waschromatographed to give a white solid.

5. Preparation of PB2,5-Ethyl-5-phenyl-2-(4-succinimidoxycarbonylbutyl)-2,4,6(1H,3H,5H)pyrimidinetrione##STR13##

This material was prepared using the procedure of step 7, preparatoryexample 4 except starting with the acid of step 2. The materialcrystallizes from ethyl ether/ethyl acetate (1:1) to give a white solid.

6. Preparation of PB3,5-Ethyl-5-phenyl-1-{4-[2-(3-succinimidoxycarbonylpropionyloxy)ethylaminocarbonyl]butyl}-2,4,6(1H,3H,5H)-pyrimidinetrione

Step 1: Preparation of5-Ethyl-1-[4-(2-hydroxyethylaminocarbonyl)butyl]-5-phenyl-2,4,6(1H,3H,5H)pyrimidinetrione

This material was prepared as outlined in step 4 of preparatory example4 except using 2-hydroxyethylamine in place of the benzyl1-piperazinecarboxylate.

Step 2: Preparation of1-{4-[2-(3-Carboxypropionyloxy)ethylaminocarbonyl]butyl}-5-ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione

A mixture of the product from step 1 (2.9 g, 0.007 mole), succinicanhydride (0.7 g, 0.007 mole), and dimethylaminopyridine (0.9 g, 0.007mole) in chloroform (100 mL) was heated with hot water (50°-60° C.) for30 min and then stirred at room temperature for 3 days. Dichloromethane(300 mL) was added, and the mixture was washed with 10% hydrochloricacid solution (2×100 mL), washed with saturated sodium chloride solution(100 mL), dried over anhydrous magnesium sulfate, filtered, and solventremoved to give an oil which was used directly in the next step.

Step 3: Preparation of PB3,5-Ethyl-5-phenyl-1-{4-[2-(3-succinimidoxycarbonylpropionyloxy)ethylaminocarbonyl]butyl)-2,4,6(1H,3H,5H)-pyrimidinetrione##STR14##

This material was prepared using the procedure outlined in step 7 ofpreparatory example 4 starting with the acid of step 2 of this example.

7. Preparation of PB4,5-Ethyl-5-phenyl-1-{4-[3-(3-succinimidoxycarbonylpropionamido)propylaminocarbonyl]butyl}-2,4,6(1H,3H,5H)-pyrimidinetrione

Step 1: Preparation of1-[4-(3-Benzyloxycarbonylaminopropylaminocarbonyl)butyl]-5-ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione

This material was prepared using the procedure outlined in step 4,preparatory example 4, except substitutingN-benzyloxycarbonyl-1,3-propanediamine for the benzyl1-piperazinecarboxylate, and the crude material was used in the nextstep.

Step 2: Preparation of1-[4-(3-Aminopropylaminocarbonyl)butyl]-5-ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrioneHydrobromide

This material was prepared as in step 5, preparatory example 4 (exceptstarting with the amide of step 1 of this example to give an oil whenpoured into ethyl ether.

Step 3: Preparation of1-{4-[3-(3-Carboxypropionamido)propylaminocarbonyl]butyl)-5-Ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione

This material was prepared by the procedure of step 6, preparatoryexample 4, except starting with the amine from step 2 of this example togive the acid.

Step 4: Preparation of PB4,5-Ethyl-5-phenyl-1-{4-[3-(3-succinimidoxycarbonylpropionamido)-propylaminocarbonyl]butyl}-2,4,6(1H,3H,5H)-pyrimidinetrione##STR15##

This material was prepared using the procedure of step 7, preparatoryexample 4 except starting with the acid of step 3 of this example.

8. Preparation of PB5,5-Ethyl-5-phenyl-1-{4-[6-(3-succinimidoxycarbonylpropionamido)-hexylaminocarbonyl]butyl}-2,4,6(1H,3H,5H)-pyrimidinetrione##STR16##

This compound was prepared by the sequence of reactions of preparationexample 7 except substituting N-benzyloxycarbonyl-1,6-hexanediamine forthe benzyloxycarbonyl-1,3 propanediamine in step 1, and the respectivereaction products thereafter in steps 2, 3, and 4 of preparation example7.

Labeled Drug Hapten Analogoues

We have prepared new labeled drug hapten analogues of the above preparedbarbiturate and hydantoin analogues which are useful in competitiveimmunoassays for barbiturates and hydantoin drugs, particularlyphenobarbital and phenytoin. The labels are those commonly used inimmunoassays having an amine or sulfhydryl group commonly used withanalytes or analyte analogs in competitive immunoassays such as enzymes,visible dyes, leuco dyes, fluorescent dyes, radioactive materials, etc.

Useful labels are enzymes such as alkaline phosphatase (ALP), glucoseoxidase (GOD) and horseradish peroxidase (HRP).

The labeled drug hapten analogues are prepared with a new processcomprising the steps of:

1) contacting a label having a nucleophilic group thereon such as anamine or sulfhydryl group, with an excess of a barbiturate or hydantoindrug hapten analogue described supra. Preferably the analogues and thelabel are dissolved in a water miscible organic solvent such asN,N-dimethylformamide, dimethyl sulfoxide (DMSO) or mixture of solventand water (buffered) before mixing together; and

2) removing the unused active ester and condensation by-products,preferably by dialysis.

The examples provided hereinafter illustrate the preparation of the newlabeled analogues of this invention. The labeled analogues were preparedusing phenytoin and phenobarbital drug hapten analogues.

Labeled Hydantoin Analogues

1. Preparation of an Amine Enriched HRP labeled hydantoin HD 1(containing an extended linker, label AHRP-HD 1, Label A).

HD 1 was dissolved in 1.452 mL dry DMF containing 10 mM4'-hydroxyacetanilide (DMF 4'-HA).

Amine-enriched HRP was prepared as described in U.S. Ser. No. 540,428,filed Jun. 18, 1990 in the name of Susan J. Danielson and Donald P.Specht, entitled "Amine-Enriched Proteins", the contents of which areexpressly incorporated herein by reference. Briefly, dry HRP wasdissolved in 0.1M MES buffer, pH 5.5, to achieve a final concentrationof 2.5×10⁻⁶ mol (100 mg) in 10 mL of buffer(MES=2-(N-morpholino)ethanesulfonic acid). The protein concentration wasdetermined by A₄₀₃ measurement using the conversion factor A₄₀₃ 1mg/mL=2.24. The HRP solution was combined with 1.5×10⁻³ mol (275 mg) ofL-lysine monohydrochloride dissolved in 10 mL of 0.1M MES buffer at pH5.5. A solution of freshly prepared1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 5×10⁻⁴mol, 960 mL) in MES buffer was added. The container was capped and mixedovernight at room temperature. The reaction was dialyzed against 0.02MMOPS buffer at pH 7.0 (3 L at 10° C.). The dialysis buffer was changed3×. MOPS=3-(N-morpholino)propanesulfonic acid.

Prior to reaction, a sample of the amine-enriched HRP was exchanged fromMOPS buffer into 0.1M EPPS buffer, pH 8.0, using 30,000 NMWL (nominalmolecular weight limit cutoff) Centricells centrifugal ultrafilters.This sample was then diluted to obtain a solution with a finalconcentration of 10 mg/mL.

The amine-enriched HRP (AHRP) (1 mL) was combined with 500 μL of a 10 mMsolution of 4'-hydroxyacetanilide in dimethylformamide (DMF 4'-HA) withvortex mixing and was then placed in a 42° C. water bath. An HD 1solution prepared by dissolving 21 mg of HD 1 in 1.452 mL of dry DMF4'-HA solution (500 μL) was added dropwise to the AHRP with vortexmixing so that the molar ratio of phenytoin/HRP was 50/1. The reactionwas incubated for 1 hour in a 42° C. water bath with gentle shaking.

The reaction was placed in Spectrapor #2 dialysis tubing along with anadditional 0.5 mL of DMF 4'-HA/0.1M EPPS (1:1) used to rinse thereaction container.

The reaction mixture was dialyzed as follows:

a) 1 L DMF 4'-HA/0.1M EPPS. pH 8.0, (1:1) at 42° C. for 1 hr;

b) Dialysis condition a) was repeated 1 time;

c) 2 L 0.1M EPPS, pH 8.0, containing 0.1% BSA at 8° C. for 15 hrs

d) 2 L of 0.1M EPPS, pH 8.0, at 8° C. for 3 hours;

e) 3 L of 0.04M tris(hydroxymethyl)aminomethane hydrochloride (TrisHCl)/0.15M NaCl, PH 7.5, at 8° C. for 3 hours; and

f) Dialysis condition e) was repeated 1 time for 3 hrs;

Following dialysis, merthiolate was added to 0.02% as a preservative,and the AHRP-HD 1 was stored refrigerated.

2. Preparation of an Amine Enriched HRP labeled HD 2 (containing anextended linker with an amide bond, label AHRP-HD 2, Label B).

HD 2 (15.5 mg) was dissolved in 1.031 mL dry DMF containing 10 mM4'-hydroxyacetanilide (DMF 4'-HA).

A solution of AHRP (10 mg/mL, 1 mL in 0.1M EPPS, pH 8.0), prepared asdescribed in label Preparatory example 1, was combined with 500 μL ofDMF 4'-HA with vortex mixing and was then placed in a 42° C. water bath.The HD 2 solution described above (500 μL) was added dropwise to theAHRP with vortex mixing so that the molar ratio was 50/1. The reactionwas incubated for 1 hour in a 42° C. water bath with gentle shaking.

The reaction product was placed in Spectrapor #2 dialysis tubing anddialyzed as follows:

a) 1 L DMF 4'-HA/0.1M EPPS, pH 8.0 (1:1) at 42° C. for 1 hr;

b) Dialysis condition a) was repeated 1 time;

c) 1.5 L 0.1M EPPS, pH 8.0, containing 0.1% bovine serum albumin (BSA)at 8° C. for 1.5 hr;

d) 1.5 L 0.1M EPPS, pH 8.0, at 8° C. for 18 hrs;

e) 1.5 L 0.04M Tris HCl/0.15M NaCl, pH 7.5, at 8° C. for 2 hrs; and

f) Dialysis condition e) was repeated 1 time for 4 hrs.

Following dialysis, 0.02% merthiolate was added as a preservative. Thelabeled hydantoin derivative was stored in a refrigerator.

3. Preparation of AHRP-HD 3 (containing an extended linker with an amidebond label AHRP-HD 3, Label C).

HD 3 (9.2 mg) was dissolved in 1 mL dry DMF containing 10 mM4'-hydroxyacetanilide (DMF 4'-HA).

A solution of AHRP, prepared as described in label preparatory example1, was dialyzed into 0.1M EPPS buffer, pH 8.0. The final concentrationwas determined to be 5.71 mg/ml.

The AHRP (1 mL) was combined with 500 μL of DMF 4'-HA with vortex mixingand was then placed in a 42° C. water bath. The HD 3 solution describedabove (500 μL) was added to the AHRP dropwise with vortex mixing so thatthe molar ratio of HD 3/AHRP was 50/1. The reaction was incubated for 1hour in a 42° C. water bath with gentle shaking. The reaction was placedin Spectrapor #2 dialysis tubing along with an additional 0.5 mL of DMF4'-HA/0.1M EPPS (1:1) used to rinse the reaction container.

The reaction was dialyzed as follows:

a) 1 LDMF 4'-HA/0.1M EPPS, pH 8.0, (1:1) at 42° C. for 1 hour;

b) dialysis condition a) was repeated 1 time;

c) 1.5 L 0.1M EPPS, pH 8.0, containing 0.1% BSA at 5° C. for 15 hours;

d) 1.5 L 0.1M EPPS, pH 8.0, for 8 hours;

e) 2 L 0.02M 3-morpholinopropanesulfonic acid (MOPS), pH 7.0. at 5° C.for 13 hours; and

f) Dialysis condition e) was repeated 2 times.

Following dialysis, merthiolate was added to a concentration of 0.02% asa preservative, and the label was stored refrigerated.

Labeled Barbiturate Drug Hapten Analogues

The following examples demonstrate the preparation of labeledbarbiturate drug hapten analogues

4. Preparation of Amine Enriched HRP labeled PB 2; label AHRP-PB 2.Label D

PB 2 was dissolved in DMSO to yield a 10.7 mg/mL solution (1.25×10⁻² M).Then 500 μL of this solution was added to amine enriched HRP/DMSOsolution (prepared similar to AHRP/DMF) dropwise while vortex mixing.The molar ratio of the phenobarbital/HRP was 50/1.

Incubation was performed at room temperature for 4 hours with shaking at2400 rpm. The sample was transferred to Spectrapor #2 dialysis tubingalong with an additional 1 mL of dialysate to rinse the reactioncontainer. The label was dialyzed into 0.02M MOPS buffer, pH 7.0, at5°-10° C. This dialysis condition was repeated three times with 2-3 L ofbuffer each time. Following dialysis, 0.02% merthiolate was added as apreservative, and the label was stored refrigerated.

5. Preparation of Amine Enriched HRP-PB 3; (AHRP-PB3 Label E containingan Extended Linker)

Amine-enriched HRP was exchanged from MOPS buffer into 0.1M EPPS buffer,pH 8.0, using a Centricell centrifugal ultrafilter (30,000 nominalmolecular weight limit). This sample was then diluted to 4.6 mL, 0.743mg/ml.

One mL of the HRP was added to a small vial (1.85×10⁻⁵ M). 500 μL ofdimethylformamide, Aldrich 22,705-6, containing 10 mM4'-hydroxyacetanilide (DMF 4'-HA) was added to the vial, vortexed, andplaced in a 42° C. water bath.

Meanwhile, PB-3 was dissolved in DMF 4'-HA to yield a 2.12 mg/mlsolution (3.70×10⁻³ M). 500 μL of this solution was added to the HRP/DMF4'-HA solution dropwise while vortex mixing. The molar ratio of thephenobarbital/HRP was 100/1.

Incubation was performed at 42° C. for 1 hour with gentle shaking in awater bath. The sample was transferred to Spectrapor #2 dialysis tubingalong with an additional 1 mL of DMF 4'-HA/0.1M EPPS (1:1) used to rinsethe reaction container. The reaction was dialyzed as follows:

a) 1 L DMF 4'-HA/0.1M EPPS, pH 8.0 (1:1) at 42° C. for 1 hr;

b) Dialysis condition a) was repeated once;

c) 1.5 L 0.1M EPPS, pH 8.0, containing 0.1% bovine serum albumin (BSA)at 5° C. overnight;

d) 1.5 L 0.1M EPPS, PH 8.0, at 5° C., 8 hrs;

e) 2.0 L 0.02M MOPS, pH 7.0 at 5° C., for at least 8 hrs; and

f) Dialysis condition e) was repeated twice.

Following dialysis, 0.02% merthiolate was added as a preservative, andthe label was stored refrigerated.

6. Preparation of an Amine Enriched HRP-PB 1; (Label AHRP-PB 1, Label F,an Active Ester of a Phenobarbital Hapten analogue Containing anExtended Linker with an Amide Bond)

Amine-enriched HRP was prepared and exchanged into 0.1M EPPS buffer, pH8.0, to yield a, 10 mg/mL solution (2.5×10-4M). Label F was made using 5mL (50 mg) amine-enriched HRP. 2.5 mL DMSO was added slowly, whilestirring over a magnetic stir plate. The solution was stirred for 15minutes at room temperature.

Meanwhile, PB 1 was dissolved in DMSO to yield a 14.9 mg/ml solution.2.5 mL of this solution was added to the HRP/DMSO solution slowly whilestirring. The molar ratio of the phenobarbital/HRP was 50/1.

Incubation was performed at room temperature for 5 hours with shaking at2400 rpm. The sample was transferred to Spectropor #2 dialysis tubingalong with additional dialysate to rinse the reaction container. Thelabel was dialyzed into 0.02M MOPS buffer, pH 7.0, at 5°-10° C. Thisdialysis condition was repeated three times with 3 L of buffer eachtime. Following dialysis, 0.02% merthiolate was added as a preservative,and the labels were stored refrigerated.

Immunocompetence

The following tests demonstrate the immunocompetence of the labels 1-6,supra.

1. Immunocompetency of label AHRP-HD 1 (Label A).

In this example, the ability of several immobilized antibodies (DilAs₈,DilAs₉, DilAs₁₄, DilAs₁₆, and DilAs₂₁) to bind AHRP-HD ₁ (label A) fromlabel preparatory example 1) is examined.

(a) Polymer bead samples, each sample having one of the above-identifiedtypes of antibodies covalently bound thereto were prepared using methodsand materials as described in U.S. Ser No. 081,206 filed Aug. 3, 1987(published EPA 88 307172.2).

(b) The ability of the immobilized antibodies to bind AHRP-HD 1 (labelA) was determined as follows:

Each of the various antibody beads were serially diluted with PBScontaining 1% BSA to give concentrations between 500 and 0.50 nMantibody binding sites. The bead dilutions were mixed with equal volumesof the label at 10×10⁻¹¹ M. Following a 1 hour incubation, the beadswere pelleted by centrifugation. A sample (100 μL) of the supernatantwas mixed with 100 μL of substrate (o-phenylenediamine/H₂ O₂). The ratesof color development at 450 nm were compared with those of standards tocalculate the amount of phenytoin-HRP label remaining in solution. Theamount of label bound to immobilized antibody at the highest antibodyconcentration tested (250 nM binding sites) is reported.

    ______________________________________                                        % Label Bound at 250 nM Antibody Binding Sites                                          Label A                                                                       (Extended Linker)                                                   ______________________________________                                        DilAs.sub.8 93                                                                DilAs.sub.9 94                                                                DilAs.sub.14                                                                              90                                                                DilAs.sub.16                                                                              96                                                                DilAs.sub.21                                                                              97                                                                ______________________________________                                    

These results show that these antibodies recognize very well AHRP-HD 1Labels (label A).

2. Hydrolytic stability of AHRP-HD 2 (label B)

This example illustrates the hydrolytic stability of a labeled phenytoinderivative of the invention having an amide bond in the linking chainbetween the label and the phenytoin nucleus AHRP-HD 2, (label B).

Beads having immobilized Kallestad antibodies were prepared as describedin U.S. Ser. No. 081,206, filed Aug. 3, 1987 (published EPA 88307172.2).

AHRP-HD 2 (label B) was diluted to 1×10⁻¹⁰ M in PBS containing 1% BSAadjusted to pH 7.3 or 8.5. The label was incubated at room temperaturefor 6 days. The label was tested for binding by immobilized antibodyafter 2 days and 6 days as follows:

Kallestad 52-2 antibody beads were serially diluted with PBS containing1% BSA to give concentrations between 500 and 0.50 nM antibody bindingsites. The bead dilutions were mixed with equal volumes of labels at10×10⁻¹¹ M. Following a 1 hour incubation, the beads were pelleted bycentrifugation. A sample (100 uL) of the supernatant was mixed with 100uL of substrate (o-phenylenediamine/H₂ O₂). The rates were compared withthose of standards to calculate the amount of label remaining insolution. The amount of label bound to immobilized antibody at thehighest antibody concentration tested (250 nM binding sites) isreported.

    ______________________________________                                        Percent Label Bound at 250 nM Antibody Binding Sites                                        Label B                                                                       (Amide Bond)                                                                  pH 7.3                                                                              pH 8.5                                                    ______________________________________                                        0 days          100     --                                                    2 days          98      99                                                    6 days          99      100                                                   ______________________________________                                    

The results show that binding of AHRP-HD 2 (label B) containing theamide bond in the linking chain showed no degradation over this timeperiod. This indicates that label B will resist degradation due tohydrolysis. Such hydrolysis could cause a shift in the assay responsewith time.

3. Comparison of Phenobarbital-HRP Labels Prepared with Valerate andExtended Linkers

In this example, the ability of several immobilized antibodies (Kall1571 and PbAs9) to bind a label with the valerate linker (label D,AHRP-PB 2) and a label with an extended linker (label F, AHRP-PB 1) werecompared.

Immobilized antibody bead samples were prepared as follows:

Polymer beads (30 mg) [poly (styrene-co-p-vinylbenzyl 2-chloroethylsulfone) (95:5 molar ratio)]were dispersed in 1 mL buffer (0.1M EPPS, pH8.5) and 0.3 mg of antibody (Kall 1571 or PbAs9) was added. The totalvolume was 1.5 mL. The mixture was rotated end over end at roomtemperature for 4 hours. Then 0.3 mL of a 10% solution of BSA was added,and the supernatants were removed and analyzed for unbound antibodyusing an anti-mouse IgG. The amount of antibody bound to the surface wascalculated using ELISA. The pellets were washed 3 times with PBS, pH7.2, by resuspending in the buffer and centrifuging. The finalredispersion was in 1.8 mL of PBS; merthiolate was added to aconcentration of 0.02%, and the preparations were stored at 4° C. untiluse.

The ability of the immobilized antibodies to bind label was determinedas follows:

Each of the various antibody beads was serially diluted with PBScontaining 1% BSA to give concentrations between 200 and 0.50 nMantibody binding sites. The bead dilutions were mixed with equal volumesof phenobarbital-HRP labels at 10×10⁻¹¹ M. Following a 1-hourincubation, the beads were pelleted by centrifugation. A sample (100 μL)of the supernatant was mixed with 100 μL of substrate(o-phenylenediamine/H₂ O₂). The rates of color development at 450 nmwere compared with those of standards to calculate the amount ofphenobarbital-HRP label remaining in solution. The amount of label boundto immobilized antibody at the highest antibody concentration tested(100 nM binding sites) is reported.

    ______________________________________                                                     % Label Bound at 100 nM                                                       Antibody Binding Sites                                                        Label D                                                                              Label F                                                   ______________________________________                                        Kall 1571      41%      76%                                                   PbAs9          49%      73%                                                   ______________________________________                                    

These results indicate that these antibodies show improved recognitionof labels prepared with haptens having the extended linking group usedin labeled drug hapten analogues provided by the invention. Thisrepresents a significant advantage since the hapten with the extendedlinker allows these antibodies to be considered for development of aphenobarbital enzyme immunoassay.

The immunoassay can be carried out using any suitable label which can beattached to the defined hydantoin and barbiturate derivatives. Inaddition to the horseradish peroxidase used in preparing the foregoinglabeled drugh hapten analogues, useful labels include radioactive tags,dyes, fluorescing agents, other enzymes, enzyme substrates, enzymeinhibitors, allosteric effectors, cofactors and other known enzymemodulators. Enzymes, such as glucose oxidase, peroxidases such ashorseradish peroxidase and amine-enriched horseradish peroxidase,alkaline phosphatase and galactosidase are preferred labels.

When an enzyme label is used, the substrate for the enzyme is present inthe element or added thereto in the developing liquid. The substrate canbe added to the element prior to or simultaneously with the liquidsample, or after completion of the binding reaction. It is within theskill of the ordinary worker in clinical chemistry to determine asuitable substrate for a given label. The substrate can be a materialwhich is directly acted upon by the enzyme label, or a material that isinvolved in a series of reactions which involve enzymatic reaction ofthe label. For example, if the enzyme label is a peroxidase, thesubstrate is hydrogen peroxide. Using glucose oxidase as an example, thesubstrate glucose is generally present in the reagent layer or is addedin the developing liquid to yield about 0.01 moles/m², and preferablyfrom about 0.001 to about 0.1 mole/m². A worker skilled in the art wouldknow how to adjust the amount of a particular substrate for the amountof enzyme label used in the assay.

When certain labels are used, e.g. enzymes, cofactors, enzyme substratesor enzyme modulators, the reagent layer contains an indicatorcomposition comprising one or more reagents which provide a detectablespecies as a result of reaction of the label. Preferably, the indicatorcomposition is a colorimetric indicator composition which provides acolorimetrically detectable species as a result of enzymatic reaction ofan enzyme-labeled ligand analog with a substrate.

The indicator composition can be a single compound which produces adetectable dye upon enzymatic reaction, or a combination of reagentswhich produce the dye. For example, when glucose is used as thesubstrate and glucose oxidase as the enzyme label, the colorimetricindicator composition can include a coupler and oxidizable compoundwhich react to provide a dye. Alternatively, the composition can includea leuco dye and peroxidase or another suitable peroxidative compoundwhich generate a detectable dye as a result of the formation of hydrogenperoxide produced when glucose oxidase converts glucose to gluconicacid. Useful leuco dyes are known in the art and include those, forexample, described in U.S. Pat. No. 4,089,747 (issued May 16, 1978 toBruschi) and U.S. Ser. No. 612,509, filed May 21, 1984 by Babb et al.The particular amounts of the colorimetric indicator composition and itsvarious components are within the skill of a worker in the art.

The immunoassay can be manual or automated. In general, the amount of aligand in a liquid is determined by taking the element from a supplyroll, chip packet or other source and physically contacting a finitearea of the spreading layer with a sample of the liquid, e.g. 1 to 100μl. The finite area which is contacted is generally no more than about100 mm².

The amount of ligand is determined by passing the element through asuitable apparatus for detecting the complexed ligand analog directly orthe detectable species formed as a result of enzymatic reaction of anenzyme label and a substrate. For example, the species can be detectedwith suitable radiometric, fluorometric or spectrophotometric apparatususing generally known procedures. In an enzymatic reaction, theresulting product is determined by measuring, for example, thereflection or transmission density or fluorescence in the center of thefinite area which was contacted with the test sample. The area which ismeasured is generally from about 3 to about 5 mm in diameter forcompeting assays. The amount of ligand in the liquid sample is inverselyproportional to the amount of label measured in the center of the finitearea. In a preferred embodiment a separate developer step is required inorder to maximize separation of complexed ligand from uncomplexedligand. Generally, label measurement is carried out after from about 5to about 180 seconds after sample contact and spreading or applicationof the developing liquid.

EXAMPLES OF THE INVENTION

The following examples demonstrate the immunoassays conveniently on dryimmunoassay elements using the labeled drug hapten analgues describedabove. The examples demonstrate the invention by conducting assays forphenytoin and phenobarbital. It will be clear however that the inventionis applicable to other barbiturate and with hydantoin drug derivatives.

In the examples the assay procedures were carried out step-wiseaccording to the following protocol. Ten μL of a sample was spotted onthe top surface of a dry immunoassay element of the invention. Theelement with the now spotted sample was then incubated at 37° C. for 5minutes. After this period of incubation the element was removed fromthe incubator and developed with 10 uL of enzyme substrate solution. Forthe assays used in the examples the label is horseradish peroxidase(HRP) and the substrate used in the developing solution is about 0.3% byweight H₂ O₂. The developing solution also contains sodium phosphatebuffer (pH 6.8) 0.01M, 4'-hydroxyacetanilide (electron transfer agent)0.005M, diethylenetriaminepentaacetic acid 10 μM and a surfactant. Thedeveloping solution causes development of the detectable species. Thebound HRP-labeled ligand catalyzes the oxidation of a colorless leucodye to its colored form. Such dyes are well known in the dry analyticalelement art and will not be described in detail here. In the examplespresented herein the leuco dye is a triarylimidazole. The rate of thecatalyzed reaction is measured from the change in reflection densityover time at 37° C. Methods and means for measuring reflection densityare well known in the analytical arts.

Example 1

Use of a labeled phenytoin analogue in (AHRP-HD 1, Label A) immunoassayfor Phenytoin.

The element was prepared using known technology to have the followingstructure:

    ______________________________________                                                                Coverage                                                                      g/m.sup.2                                             ______________________________________                                        Spreading                                                                              Immobilized Kallestad 52-2                                                                          0.1                                            Layer    Antibody                                                                      Particles of poly[m-&p-vinyl-                                                                       130                                                     toluene (64:36)-co-methacrylic                                                acid] (98:2 weight ratio (30                                                  μm)                                                                        4,5-bis(4-dimethylaminophenyl)-                                                                     0.2                                                     2-(4-hydroxy-3,5-dimethoxy-                                                   phenyl)imidazole leuco dye                                                    poly(methyl acrylate-co-sodium                                                                      2.58                                                    2-acrylamido-2-methylpropane-                                                 sulfonate-co-2-acetoacetoxy-                                                  ethyl methacrylate) (90:4:6                                                   weight ratio)                                                                 N-[tris(hydroxymethyl)methyl]-                                                                      0.219                                                   2-aminoethanesulfonic acid; (pH                                               7.0)                                                                          dimethyl sulfoxide    1.8                                                     5,5-dimethyl-1,3-cyclohexane-                                                                       0.05                                                    dione                                                                         Zonyl ® FSN nonionic surfactant                                                                 0.054                                                   (DuPont)                                                             Gelatin  hardened gelatin      10                                             Layer    4'-hydroxyacetanilide 0.15                                                    N-[tris(hydroxymethyl)methyl]-                                                                      0.68                                                    2-aminoethanesulfonic acid; (pH                                               7.0)                                                                          Triton ® X-100 nonionic                                                                         0.02                                                    surfactant (Rohm & Haas)                                                      Poly(ethylene terephthalate) Support                                 ______________________________________                                    

A series of human serum based calibrators containing phenytoin and aAHRP-HD 1 (label A ) was prepared. Lyophilized serum based calibrators(BioRad) were rehydrated with 3 mL of deionized water. The concentrationof phenytoin varied from 0.5 to 128 μg/ml. Label A was added to give afinal concentration of 1 nM.

The series of phenytoin standards (10 μL aliquots) was spotted onto thespreading layers of a series of identical analytical elements having theabove configuration. After 5 minutes incubation at 37° C., a developingsolution (10 μL) comprising hydrogen peroxide (0.03%), sodium phosphatebuffer (0.01M, pH 6.8), 4'-hydroxyacetanilide (5 mM) anddiethylenetriaminepentaacetic acid (10 μM) was added to initiate dyeformation. After about 1 minute, the reflection density (D_(r)) wasmeasured at the center of the area at 680 nm at 37° C. The D_(r) valueswere converted to D_(t) by the Clapper-Williams transform. The change inD_(t) over 30 seconds was calculated. The results are shown below:

    ______________________________________                                        Phenytoin, μg/ml                                                                           Rate (D.sub.t /min)                                           ______________________________________                                        0.5             0.0954                                                        1.0             0.0950                                                        2.0             0.0869                                                        4.0             0.0788                                                        8.0             0.0661                                                        16.0            0.0522                                                        32.0            0.0364                                                        64.0            0.0237                                                        128.0           0.0157                                                        ______________________________________                                    

The results show that there is a significant change in rates over thedesired dynamic range. The therapeutic range is 10-20 μg/ml. Thisexample demonstrates an analytical element useful with the immunoassaymethod provided by the invention and its use in a competitive bindingassay to detect the drug phenytoin.

Example 2

Use of AHRP-HD 2 (Label B) having the extended linker and an amide bondin an immunoassay for phenytoin.

This example demonstrates another analytical element provided by thisinvention and its use as a labeled hydantoin derivative of the inventionin a competitive binding assay to detect the drug phenytoin.

The element was prepared using known technology to have the followingstructure:

    ______________________________________                                                                 Coverage                                                                      g/m.sup.2                                            ______________________________________                                        Spreading                                                                              Immobilized Kallestad 52-2                                                                          0.15                                           Layer    antibody                                                                      Particles of poly[m-&p-vinyl-                                                                       130                                                     toluene (64:36)-co-methacrylic                                                acid) (98:2 weight ratio) (30                                                 μm)                                                                        poly(methyl acrylate-co-sodium                                                                      0.2                                                     2-acrylamido-2-methylpropane-                                                 sulfonate-co-2-acetoacetoxy-                                                  ethyl methacrylate) (90:4:6                                                   weight ratio)                                                                 N-[tris(hydroxymethyl)methyl]-                                                                      0.219                                                   2-aminoethanesulfonic acid; (pH                                               7.0)                                                                          dimethyl sulfoxide    1.8                                                     5,5-dimethyl-1,3-cyclohexane-                                                                       0.05                                                    dione                                                                         Zonyl ® FSN nonionic surfactant                                                                 0.054                                                   (DuPont)                                                             Gelatin  hardened gelatin      10                                             Layer    4'-hydroxyacetanilide 0.15                                                    N-[tris(hydroxymethyl)methyl]-                                                                      0.68                                                    2-aminoethanesulfonic acid; (pH                                               7.0)                                                                          Triton ® X-100 nonionic                                                                         0.02                                                    surfactant (Rohm & Haas)                                                      Poly(ethylene terephthalate) Support                                 ______________________________________                                    

A series of human serum based calibrators containing phenytoin and labelB (AHRP-HD 2) was prepared. Frozen serum based calibrators containedphenytoin in concentrations from 0 to 70 μg/mL. Label B was added togive a final concentration of 1 nM.

A series of phenytoin standards (10 μL aliquots) was spotted onto thespreading layers of a series of identical analytical elements accordingto the above configuration. After 5 minutes incubation at 37° C., adeveloping solution (10 μL) con, rising hydrogen peroxide (0.03%),sodium phosphate buffer (0.01M, pH 6.8), 4'-hydroxyacetanilide (5 mM),and diethylenetriaminepentaacetic acid (10 μM) was added to initiate dyeformation. After about 1 minute, the reflection density (D_(r)) wasmeasured at the center of the area at 680 nm at 37° C. The D_(r) valueswere converted to D_(t) by the Clapper-Williams transform. The change inD_(t) over 30 seconds was calculated. The results are shown below:

    ______________________________________                                        Phenytoin, μg/mL                                                                           Rate (D.sub.t /min)                                           ______________________________________                                         0              0.0759                                                        10              0.0338                                                        20              0.0293                                                        40              0.0206                                                        70              0.0159                                                        ______________________________________                                    

The results show that there is a significant change in rates over thedesired dynamic range. The therapeutic range is 10-20 μg/ml.

Example 3

Use of labeled phenytoin derivative, AHRP-HD 3 (Label C) having anextended linker prepared from hexanediamine with amide bonds in animmunoassay for phenytoin.

This example demonstrates the use of the analytical element described inExample 2 with a different labeled hydantoin derivative of the inventionin a competitive binding assay to detect the drug phenytoin.

A series of human serum based calibrators containing phenytoin and LabelC from label preparation Example 3 was prepared. Frozen serum basedcalibrators contained phenytoin in concentrations from 0 to 70 μg/ml.Label C was added to give a final concentration of 1 nM.

A series of phenytoin standards (10 μL aliquots) was spotted onto thespreading layers of a series of analytical elements identical to thatdescribed in example 10 except containing Label C in place of Label B.After 5 minutes incubation at 37° C., a developing solution (10 μL)comprising hydrogen peroxide (0.03%), sodium phosphate buffer (0.01M, pH6.8), 4'-hydroxyacetanilide (5 mM), and diethylenetriaminepentaaceticacid (10 μM) was added to initiate dye formation. After about 1 minute,the reflection density (D_(r)) was measured at the center of the area at680 nm at 37° C. The D_(r) values were converted to D_(t) by theClapper-Williams transform. The change in D_(t) over 30 seconds wascalculated. The results are shown below:

    ______________________________________                                                 0  0.0872                                                                    10  0.0439                                                                    20  0.0340                                                                    40  0.0255                                                                    70  0.0202                                                            ______________________________________                                    

The results show that there is a significant change in rates over thedesired dynamic range.

Example 4

Use of a Phenobarbital-HRP Label Prepared with an Extended Linker in anEnzyme Immunoassay for Phenobarbital

This example demonstrates the preparation of an analytical element ofthis invention and its use in a competitive binding assay to detect thedrug phenobarbital.

The element was prepared having the following structure:

    ______________________________________                                                                 Coverage                                                                      (g/m.sup.2)                                          ______________________________________                                        Spreading                                                                              Immobilized Kallestad 1571                                                                          0.125                                          Layer    Antibody                                                                      Particles of poly[m-&p-                                                                             130                                                     vinyltoluene (64:36)-co-                                                      methacrylic acid] (92:2 weight                                                ratio) (30 μm)                                                             4,5-bis(4-dimethylaminophenyl)-2-                                                                   0.2                                                     (4-hydroxy-3,5-                                                               dimethoxyphenyl)imidazole leuco                                               dye                                                                           poly(methyl acrylate-co-sodium 2-                                                                   2.58                                                    acrylamido-2-methyl-                                                          propanesulfonate-co-2-                                                        acetoacetoxyethyl methacrylate)                                               (90:4:6 weight ratio)                                                         N-[tris(hydroxymethyl)methyl-2-                                                                     0.219                                                   aminoethanesulfonic acid; (pH                                                 7.0)                                                                          dimethyl sulfoxide    1.8                                                     5,5-dimethyl-1,3-cyclohexanedione                                                                   0.05                                                    Zonyl ® FSN nonionic surfactant                                                                 0.054                                                   (DuPont)                                                             Gelatin  hardened gelatin      10                                             Layer    4'-hydroxyacetanilide 0.15                                                    N-[tris(hydroxymethyl)methyl]-2-                                                                    0.68                                                    aminoethanesulfonic acid; (pH                                                 7.0)                                                                          Triton ™ X-100 nonionic                                                                          0.02                                                    surfactant (Rohm & Haas)                                                      Poly(ethylene                                                                 terephthalate) Support                                               ______________________________________                                    

A series of human serum based calibrators containing phenobarbital and aphenobarbital-HRP label (label AHRP-PB 3 or label E from the PB-3 oflabel preparation 6) was prepared. The concentration of phenobarbitalvaried from 0 to 80 μg/ml. The phenobarbital-HRP label was added to givea final concentration of 6 nM.

The series of phenobarbital standards (10 μL aliquots) was spotted ontothe spreading layers of a series of analytical elements. After 5 minincubation at 37° C., a wash solution (10 μL) comprising hydrogenperoxide (0.03%), sodium phosphate buffer (0.01M, pH 6.8),4'-hydroxyacetanilide (5 mM) and diethylenetriaminepentaacetic acid (10μM) was added to wash unbound complex away from the center of the areato which the phenobarbital standards had been applied, and to initiatedye formation. After about 1 minute, the reflection density (D_(r)) wasmeasured at the center of the area at 680 nm at 37° C. The D_(r) valueswere converted to D_(t) by the Clapper-Williams transform. The change inD_(t) over 30 seconds was calculated. The results are shown below:

    ______________________________________                                        Phenobarbital, μg/mL                                                                        Rate (D.sub.t /min)                                          ______________________________________                                         0               0.095                                                         7               0.073                                                        20               0.053                                                        40               0.038                                                        80               0.026                                                        ______________________________________                                    

The results show that there is a significant change in rates over thedesired dynamic range. The therapeutic range is 20-40 μg/mL.

Example 5

Use of a Phenobarbital-HRP Label Prepared with an Extended Linker withan Amide Bond in an Enzyme Immunoassay for Phenobarbital

This example demonstrates the preparation of an analytical element ofthis invention and its use in a competitive binding assay to detect thedrug phenobarbital.

The element was prepared having the following structure:

    ______________________________________                                                                 Coverage                                                                      (g/m.sup.2)                                          ______________________________________                                        Spreading                                                                              Immobilized PbAs9     0.40                                           Layer    antibody                                                                      Particles of poly[m-&p-                                                                             130                                                     vinyltoluene (64:36)-co-                                                      methacrylic acid) (92:2 weight                                                ratio) (30 μm)                                                             4,5-bis(4-dimethylaminophenyl)-                                                                     0.2                                                     2-(4-hydroxy-3,5-                                                             dimethoxyphenyl)imidazole leuco                                               dye                                                                           poly(methyl acrylate-co-sodium                                                                      2.58                                                    2-acrylamido-2-                                                               methylpropanesulfonate-co-2-                                                  acetoacetoxyethyl methacrylate)                                               (90:4:6 weight ratio)                                                         N-[tris(hydroxymethyl)methyl]-                                                                      0.219                                                   2-aminoethanesulfonic acid; (pH                                               7.0)                                                                          dimethyl sulfoxide    1.8                                                     5,5-dimethyl-1,3-     0.05                                                    cyclohexanedione                                                              Zonyl ™ FSN nonionic surfactant                                                                  0.054                                                   (DuPont)                                                             Gelatin  hardened gelatin      10                                             Layer    4'-hydroxyacetanilide 0.15                                                    N-[tris(hydroxymethyl)methyl]-                                                                      0.68                                                    2-aminoethane-sulfonic acid;                                                  (pH 7.0)                                                                      Triton ™ X-100 nonionic                                                                          0.02                                                    surfactant (Rohm & Haas)                                                      Poly(ethylene terephthalate)                                                  Support                                                              ______________________________________                                    

A series of human serum based calibrators containing phenobarbital and aphenobarbital-HRP label (AHRP-PB-1,label F from label preparation 6) wasprepared. Frozen serum based calibrators contained phenobarbital inconcentrations from 0 to 80 μg/mL. The phenobarbital-HRP label was addedto give a final concentration of 8 nM.

A series of phenobarbital standards (10 μL aliquots) was spotted ontothe spreading layers of a series of analytical elements. After 5 minincubation at 37° C., a wash solution (10 μL) comprising hydrogenperoxide (0.03%), sodium phosphate buffer (0.01M, pH 6.8),4'-hydroxyacetanilide (5 mM), and diethylenetriaminepentaacetic acid (10μM) was added to wash unbound complex away from the center of the areato which the phenobarbital standards had been applied, and to initiatedye formation. After about 1 min, the reflection density (D_(r)) wasmeasured at the center of the area at 680 nm at 37° C. The D_(r) valueswere converted to D_(t) by the Clapper-Williams transform. The change inD_(t) over 30 seconds was calculated. The results are shown below:

    ______________________________________                                        Phenobarbital, μg/mL                                                                        Rate (D.sub.t /min)                                          ______________________________________                                         0               0.116                                                         7               0.083                                                        20               0.043                                                        40               0.026                                                        80               0.017                                                        ______________________________________                                    

The results show that there is a significant change in rates over thedesired dynamic range. The therapeutic range is 20-40 μg/mL.

In the elements of the examples of the invention the labeled drug haptenderivative is spotted on the element with the sample. Alternatively thelabeled drug hapten analogue could be gravure coated over the elementspreading layer.

The invention has been described in detail with particular reference topreferred embodiments thereof, but it will be understood that variationsand modifications can be effected within the spirit and scope of theinvention.

What is claimed is:
 1. An immunoassay method for a hydantoin or abarbiturate drug comprising the steps of:A. contacting a liquid samplecontaining the drug, with a labeled analog of the drug in the presenceof immobilized antibodies for the drug under conditions that promote theformation of antibody-drug immunocomplexes; separating bound labeleddrug analog from unbound labeled drug analog; B. contacting boundlabeled drug analog or unbound labeled drug analog with a developersolution; separating bound labeled drug analog from unbound labeled druganalog; and C. determining the quantity of the drug in the liquid bymeasuring bound labeled or unbound labeled drug analog; wherein thelabeled drug analog conforms to the structure: ##STR17## wherein Arepresents a hydantoin nucleus of the structure ##STR18## or abarbiturate nucleus of the structure ##STR19## wherein R₁ eachindependently represents hydrogen, alkyl of 1 to 10 carbon atoms, orphenyl;R₂ represents C₁ to C₁₀ alkylene or such alkylene groupsinterrupted with at least one or more ester groups, amide groups --O--,--S--or --NR--, wherein R represents hydrogen or C₁ to C₆ alkyl; R₄, R₅and R₆, each independently, represent C₁ to C₁₀ alkylene or suchalkylene groups interrupted with ester groups, amide groups, --O--,--S-- or --NR--, wherein R represents hydrogen or C₁ to C₆ alkyl; R₃represents C₁ to C₆ alkylene; Z represents --O--, --S-- or --NR--,wherein R represents hydrogen or C₁ to C₆ alkyl; Label represents anenzymem is 0, 1 or 2; n is 0, 1 or 2; m +n >0; andfurther provided that(i) at least one of the R₁ groups is phenyl; and (ii) the bracketedcomponents of structure I can appear therein in any order and wherein,the linking group is other than a derivative of a saturated orunsaturated monocarboxylic acid having from 2 to 12 carbon atoms.
 2. Animmunoassay element containing a labeled drug analogue according toclaim
 1. 3. The method of claim 1 in which the labeled drug analogueconforms to the structure wherein:each R₁ independently represents ethylor phenyl; R₂ represents butylene; R₄, R₅ and R₆ each independentlyrepresents ethylene or hexylene; Z represents --O-- or --NH--; and Labelrepresents enzyme.
 4. The immunoassay of claim 1 wherein the label ishorseradish peroxidase (HRP) or amine enriched horseradish peroxidase(AHRP), the labeled drug is a labeled phenytoin or phenobarbitalanalogue and the linking group connecting the drug hapten analogue tothe horseradish peroxidase is selected from the group consistingof:tetramethylenecarbonyliminohexamethylene-iminocarbonylethylenecarbonyl,tetramethylenecarbonyl-1,4-piperazinylene-carbonylethylenecarbonyl, andtetramethylenecarbonyliminoethyleneoxy-carbonylethylenecarbonyl.
 5. Themethod of any one of claims 3, 4, or 1 when carried out on animmunoassay element.
 6. The immunoassay method of claim 1 wherein thestructure ##STR20## in the linking chain is selected from the groupconsisting of 1,4-piperazinylene; and 1,3-imidazolidinylene.
 7. Animmunoassay element having a layer, zone or coating containing a labeleddrug analog conforming to the structure: ##STR21## wherein A representsa hydantoin nucleus of the structure ##STR22## or a barbiturate nucleusof the structure ##STR23## wherein R₁ each independently representshydrogen, alkyl of 1 to 10 carbon atoms, or phenyl;R₂ represents C₁ toC₁₀ alkylene or such alkylene groups interrupted with at least one ormore ester groups, amide groups --O--, --S--, or --NR--, wherein Rrepresents hydrogen or C₁ to C₆ alkyl; R₄, R₅ and R₆, eachindependently, represents C₁ to C₁₀ alkylene or such alkylene groupsinterrupted with ester groups, amide groups, --O--, --S--, or --NR--,wherein R represents hydrogen or C₁ to C₆ alkyl; R₃ represents C₁ to C₃alkylene; Z represents --O--, --S--, or --NR--, wherein R representshydrogen or C₁ to C₆ alkyl; Label represents an enzymem is 0, 1, or 2; nis 0, 1, or 2; m +n >0;provided that (i) at least one of the R₁ groupsis phenyl; and (ii) the bracketed components of structure I can appeartherein in any order and wherein the linking group is other than aderivative of a saturated or unsaturated monocarboxylic acid having from2 to 12 carbon atoms.